Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin:potential therapy for burn wounds

BACKGROUND Impaired wound healing can be associated with different pathological states.Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide.Mesenchymal stem cells(MSCs)possess the ability to regenerate tissues by secreting factors involved in promoting cell migration,proliferation and differentiation,while suppressing immune reactions.Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIM To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODS Human umbilical cord MSCs(hU-MSCs)were isolated and characterized by surface markers,CD105,vimentin and CD90.For preconditioning,hU-MSCs were treated with isorhamnetin after selection of the optimized concentration(5μmol/L)by cytotoxicity analysis.The migration potential of these MSCs was analyzed by the in vitro scratch assay.The healing potential of normal,and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound.Normal,and preconditioned MSCs(IH+MSCs)were transplanted after 72 h of burn injury and observed for 2 wk.Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTS The scratch assay analysis showed a significant reduction in the scratch area in the case of IH+MSCs compared to the normal untreated MSCs at 24 h,while complete closure of the scratch area was observed at 48 h.Histological analysis showed reduced inflammation,completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH+MSCs.Gene expression analysis was time dependent and more pronounced in the case of IH+MSCs.Interleukin(IL)-1β,IL-6 and Bcl-2 associated X genes showed significant downregulation,while transforming growth factorβ,vascular endothelial growth factor,Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound,showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSION Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation,and improving tissue architecture and wound healing.The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.

Effect of Different Isorhamnetin of Different Density Cultured Rat C6 Glioma Cells in Vitro

Objective To explore the influence of different concentrations of isorhamnetin on C6 glioma cell morphology.Methods Set the blank control group,blank solvent control group and reagent group of four concentration,the growth of cells were observed under microscope;MTT assay was used to test the effect of isorhamnetin on cultured C6 glioma cells,as well as calculate the cell inhibition rate and survival rate;flow cytometry was used to check the detection peak and detection rate of Isorhamnetin group and negative control apoptosis group,and analyzed the relationship between different concentrations of isorhamnetin and C6 glioma cell apoptosis rate;total protein was extracted from cells,and used Western blotting to detected total AKT protein and Ser473 AKT protein loci in cells;used SD rats to construct brain glioma model,feed isorhamnetin plain to them for five days,and then used HPLC to detect plasma,liver,brain tissue content.Results Under the observation of inverted microscope and image analysis,after using Isorhamnetin,tumor cells appear apoptosis and necrosis change.Display with different Isorhamnetin MTT colorimetric method shows that the higher the concentration of added Isorhamnetin,the worse the growth rate of C6 glioma cells in vitro,and the higher the Inhibitory rate,the lower survival rate.The flow cytometric detection shows the C6 glioma cells which is added 40 ug/ul Isorhamnetin have the highest rate of apoptosis.After adding 80μg/μl concentration of the isorhamnetin,C6 glioma cells have the lowest survival rate.Western blot test shows the AKT protein and Ser473 total site AKT protein density is in reverse proportion to the increase of the concentration of the isorhamnetin.High performance liquid chromatographic method has determined that there are isorhamnetin in both the rat plasma and brain tissue,which shows that the plasma and tissue all have different isorhamnetin distribution,and isorhamnetin mainly exist in the brain tissue.Conclusion Low concentration of isorhamnetin can induce apoptosis of C6 glioma cells,and high concentration of isorhamnetin can lead to apoptosis and necrosis of C6 glioma cells in vitro,which has obvious inhibitory effect on the growth of glioma cells,and the mechanism is closely related to PI3K/AKT pathway,and in SD rat brain glioma model,the high performance liquid chromatography was used to detect the content of plasma and brain tissue,which indicated the isorhamnetin has target in brain tissue,which provided experimental evidence for the development and utilization of isorhamnetin in mice.

Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway

Objective To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis(RA).Methods Tumor necrosis factor(TNF)-α-induced fibroblast-like synoviocytes(FLS)was exposed to additional isorhamnetin(10,20 and 40µmol/L).Overexpression vectors for matrix metalloproteinase-2(MMP2)or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function.RA-FLS viability,migration,and invasion were evaluated.Moreover,a collagen-induced arthritis(CIA)rat model was established.Rats were randomly divided to sham,CIA,low-,medium-,and high-dosage groups using a random number table(n=5 in each group)and administed with normal saline or additional isorhamnetin[2,10,and 20 mg/(kg·day)]for 4 weeks,respectively.Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats.The levels of MMP2,MMP9,TNF-α,interleukin-6(IL-6),and IL-1β,as well as the phosphorylation levels of SRC,extracellular regulated kinase(ERK),and cyclic adenosine monophosphate response element-binding(CREB),were detected in RA-FLS and synovial tissue.Molecular docking was also used to analyze the binding of isorhamnetin to SRC.Results In in vitro studies,isorhamnetin inhibited RA-FLS viability,migration and invasion(P<0.05).Isorhamnetin downregulated the levels of MMP2,MMP9,TNF-α,IL-6,and IL-1βin RA-FLS(P<0.05).The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion,as well as the levels of TNF-α,IL-6,and IL-1β(P<0.05).Furthermore,isorhamnetin bound to SRC and reduced the phosphorylation of SRC,ERK,and CREB(P<0.05).SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability,migration and invasion,as well as the negative regulation of MMP2 and MMP9(P<0.05).In in vivo studies,isorhamnetin decreased arthritis index scores(P<0.05)and alleviated synovial inflammation.Isorhamnetin reduced the levels of MMP2,MMP9,TNF-α,IL-6,and IL-1β,as well as the phosphorylation of SRC,ERK,and CREB in synovial tissue(P<0.05).Notably,the inhibitory effect of isorhamnetin was more pronounced at higher concentrations(P<0.05).Conclusion Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways,suggesting that isorhamnetin may be a potential therapeutic agent for RA.

岷山红三叶草异黄酮对运动大鼠免疫功能及运动能力的影响

目的:研究岷山红三叶草异黄酮对大鼠免疫功能及运动能力的影响。方法:将40只大鼠随机均分为四组:安静组、安静给药组、运动组和运动给药组,建立递增运动负荷大鼠模型,测量运动大鼠力竭时间、免疫脏器指数、大鼠免疫球蛋白及T淋巴细胞亚群含量。结果:经过6周的大强度运动,服用岷山红三叶草异黄酮大鼠组与相应的对照组比较,其力竭运动时间明显延长,免疫脏器指数和T淋巴细胞亚群(CD_(4)^(++)、CD_(8)^(+)、CD_(4)^(+)/CD_(8)^(+))、免疫球蛋白(IgA、IgG、IgM)的含量均有明显上升和变化。结论:(1)岷山红三叶草异黄酮可明显提高大强度耐力训练大鼠力竭运动时间,提高运动能力;(2)岷山红三叶草异黄酮对运动大鼠的免疫系统产生影响,可以通过调节免疫细胞的功能,增强机体的免疫能力,并且对免疫抑制有一定改善。

Effects of ginkgo flavone aglycone on atherosclerosis based on network pharmacology,molecular docking,and in vitro experiments

Background:Ginkgo flavone aglycones(GA),a Ginkgo(Ginkgo biloba)extract,has been proven to have good biological activity in atherosclerosis(AS)treatment.Moreover,its active compounds and the corresponding mechanism for the treatment of AS remain unclear.Methods:To evaluate and identify the potential pharmacological mechanisms of GA in AS treatment,the program Cytoscape was used to generate network mappings of the GA-AS-potential target gene.GO and KEGG enrichment analyses were performed to further investigate the potential mechanism of AS and the pharmacological properties of GA.A molecular docking approach was utilized to determine the GA components that interact with Akt.In vitro experiments were carried out to identify the anti-atherosclerotic effects of GA by targeting Akt.Results:Network pharmacological research determined that the active components of GA(quercetin,kaempferol,and isorhamnetin)correlated with AS target genes such as AKT1,EGFR,SRC,ESR1,PTGS2,MMP9,KDR,GSK3B,APP,and MMP2,respectively.GO enrichment and KEGG analysis showed that PI3K-Akt signaling may play an important role in GA treatment.Molecular docking experiments indicated that quercetin,kaempferol,and isorhamnetin integrate into the binding pockets of the most potentially beneficial GA-AS target protein(Akt).Consequently,cell experiments were conducted to support the anti-atherosclerotic activity of GA on AS by inhibiting the phosphorylation of AKT1 and its downstream signaling molecules,which regulated the proliferation of HASMCs.Conclusion:Our results detailed GA's active ingredients,potential targets,and molecular basis against AS.GA may exert anti-atherosclerotic effects by suppressing Akt phosphorylation and inhibiting the proliferation of HASMCs.It also proposed a viable approach to determining the scientific foundation and therapeutic mechanism of Chinese herbal medicine extracts in disease therapy.

基于薄层色谱与一测多评法的沙棘颗粒质量评价方法研究

目的:优化中成药沙棘颗粒的薄层鉴别方法,建立多成分高效液相色谱一测多评方法。方法:采用薄层色谱法对槲皮素、异鼠李素和山柰素进行定性鉴别;采用高效液相色谱法,C18色谱柱(250 mm×4.6 mm,5µm),流动相为乙腈-0.1%磷酸水溶液,梯度洗脱,流速为1.0 mL·min^(–1),柱温为35℃,检测波长为365 nm,以槲皮素为内参物计算异鼠李素和山柰素含量。结果:薄层色谱鉴别方法斑点清晰、专属性好;与外标法对比,一测多评法准确度高、耐用性好。结论:优化的薄层色谱法与建立的一测多评法可靠、准确、重复性好,可为沙棘颗粒的质量标准提升提供参考。

异鼠李素对人卵巢癌作用机制

目的 基于网络药理学和细胞实验探求异鼠李素治疗卵巢癌的作用机制。方法 文献检索收集异鼠李素的作用靶点,通过Swiss Target Prediction数据库预测靶点,下载疾病基因及找到药物及与疾病靶点的交集,进行蛋白质-蛋白质相互作用分析,用Cytoscape软件得到核心基因,进行富集分析及分子对接。体外培养人卵巢癌SKOV3细胞,用不同浓度的异鼠李素(0、10、25 mg/L)处理细胞,比较各组细胞抑制率,并进行AKT、p-AKT、Bcl2、BAX蛋白表达比较。结果 异鼠李素作用于卵巢癌的96个潜在靶点及14个核心基因。富集分析结果提示,作用靶点与丝氨酸/苏氨酸蛋白激酶有关,主要富集在PI3K/AKT通路,分子对接提示异鼠李素与各靶点具有良好的结合活性。体外实验证实异鼠李素可以抑制卵巢癌细胞的活性,流式细胞检测发现异鼠李素处理组凋亡率增加,Western blot结果显示,异鼠李素可以降低p-AKT/AKT蛋白表达比率、降低Bcl2及升高BAX蛋白的表达,从而发挥抗肿瘤作用。结论 异鼠李素可以促进卵巢癌细胞的凋亡。

响应面优化提取新鲜沙棘果中异鼠李素及其两性聚合物结构修饰

目的:建立响应面优化提取新鲜沙棘果中异鼠李素的方法,采用高分子两性聚合物修饰异鼠李素。方法:采用Design-Expert 8.0.6软件及其Box-Behnken优化从新鲜沙棘果中提取异鼠李素,制备的异鼠李素采用自组装模式制成两性聚合物。结果:优化的提取温度为75℃,提取时间60 min,100 g新鲜沙棘果中盐酸加入量35 mL;采用自组装模式制成异鼠李素两性聚合物反应完全,溶解度良好。结论:响应面法稳定可行,可用于新鲜沙棘果中异鼠李素工艺优化;自组装模式制成异鼠李素两性聚合物结构修饰可行。

HPLC法测定蒙药沙蓬茎、叶、花中槲皮素和异鼠李素的含量

目的建立蒙药沙蓬茎、叶、花中槲皮素和异鼠李素的含量,分析槲皮素和异鼠李素在沙蓬不同部位的分布情况。方法采用高效液相色谱法(HPLC)测定槲皮素和异鼠李素的含量。结果槲皮素和异鼠李素分别在0.0107~0.5336μg、0.0421~2.1038μg范围内与峰面积具有很好的线性关系,回收率分别为100.06%、102.59%,相对标准偏差(RSD)分别为1.52%、1.67%(n=6),沙蓬茎、叶、花中槲皮素和异鼠李素的总量依次为叶>花>茎。结论该方法精密度高,分离度好,可用于蒙药沙蓬茎、叶、花中槲皮素和异鼠李素的含量测定。

异鼠李素通过nSMase2依赖的外泌体生成途径抑制结肠癌肝转移的作用机制初探

目的探讨中药活性成分异鼠李素(isorhamnetin,ISO)对结肠癌肝转移和肿瘤外泌体分泌的干预作用,并进一步揭示其潜在作用机制。方法使用CCK-8法检测ISO对小鼠结肠癌细胞MC38细胞增殖的影响。使用NanoSightNS300系统检测ISO对MC38细胞外泌体释放的影响。通过实时荧光定量检测系统(quantitative PCR detecting system,qPCR)检测ISO对MC38细胞对中性鞘磷脂酶-2(neutral sphingomyelinase 2,nSMase2)、Rab27a等外泌体生成、分泌相关基因表达的影响。将12只C57BL/6小鼠随机分为对照组、MC38-外泌体组、ISO组、阳性对照组(外泌体抑制剂GW4869组),造模4周后检测小鼠肝脏转移灶数量及肝脏重量。通过qPCR检测肝转移灶中nSMase2、Rab27a等外泌体生成、分泌相关基因的表达情况。结果ISO能够抑制MC38细胞增殖,且在20μmol/L浓度以下时,抑制率低于10%,为无毒剂量;ISO能够减少MC38细胞外泌体的分泌,且具有时间依赖性(P<0.05);ISO能够抑制MC38细胞中外泌体生成相关基因nSMase2 mRNA的表达(P<0.001),而对外泌体分泌相关基因表达无明显抑制作用。与对照组比较,ISO组小鼠的肝脏重量明显降低(P<0.05),转移瘤数目明显减少;ISO能明显抑制小鼠结肠癌肝转移灶中外泌体生成相关基因nSMase2 mRNA的表达(P<0.001)。结论ISO可能通过nSMase2依赖途径抑制小鼠结肠癌细胞外泌体生成,进而抑制结肠癌肝转移。

植物乳杆菌发酵对沙棘果汁中苦味物质的转化作用

目的:改善沙棘果汁口感,去除涩味物质异鼠李素糖苷。方法:用植物乳杆菌发酵沙棘果汁,研究益生菌的生长曲线,总酚含量和抗氧化活性等各项指标在发酵过程中的变化以及异鼠李素糖苷和异鼠李素含量的变化。结果:植物乳杆菌67-25-2发酵得到的沙棘果汁其涩味得到改善,感官评分达到85分,发酵后提高了果汁的总游离酚含量和抗氧化活性,在62 h时达到最高值,其中总酚含量提高至598.9 mg GAE/L,ABTS^(+)自由基和DPPH自由基清除率分别达到67.5%和61.8%。皮尔逊相关性分析发现,总酚含量、异鼠李素含量与DPPH自由基清除率、ABTS^(+)自由基清除率显著正相关(P<0.01)。结论:植物乳杆菌67-25-2发酵后显著(P<0.05)减少了沙棘中主要的涩味物质(异鼠李素-3-O-新橙皮苷),释放出活性更强的黄酮苷元类物质(异鼠李素),显著(P<0.05)提高了沙棘果汁的总酚含量和抗氧化活性。该研究可为改善沙棘果实口感,同时为开发含有高活性转化产物的功能型发酵沙棘食品奠定理论基础。

Isorhamnetin protects zearalenone-induced damage via the PI3K/Akt signaling pathway in porcine ovarian granulosa cells

Zearalenone(ZEA)is widely derived from moldy cereal grain,which has adverse effects on animal reproduction.In particular,pigs are more sensitive to ZEA-induced toxicity than other animals.Isorhamnetin has extensive pharmacological activity.However,the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear.This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism.Two experiments were conducted,where a minimum of 3 biological replicates were used for each treatment.In Exp.1,ovarian granulosa cells were treated with different concentrations of isorhamnetin(1,5,10,20 and 30μmol/L)and ZEA(0,10,30,60,90 and 120μmol/L)for 24 h.Our results indicated that 60μmol/L ZEA(half-maximal inhibitory concentration value)and 20μmol/L isorhamnetin(the most effective concentration against ZEA-induced cytotoxicity)were optimum concentrations.In Exp.2,ovarian granulosa cells were treated with isorhamnetin(20μmol/L)for 2 h,before treatment with ZEA(60μmol/L)for 24 h.Apoptosis,endoplasmic reticulum stress,oxidative stress,proliferation and hormone secretion of ovarian granulosa cells were detected.Our findings showed that isorhamnetin suppressed(P<0.05)ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins(B-cell lymphoma-2[Bcl-2],Bcl2-associated x[Bax]and cleaved caspase-3[C-Casp3]).Changes in intracellular Ca2+levels and C/EBP homologous protein(CHOP),recombinant activating transcription factor 6(ATF6),glucose regulated protein78 k D(GRP78)indicated that isorhamnetin rescued(P<0.05)ZEA-induced endoplasmic reticulum stress.Furthermore,isorhamnetin prevented(P<0.05)ZEA-induced oxidative stress via the mitogen-activated protein kinase(P38)signaling pathway.Mechanistically,isorhamnetin stimulated(P<0.05)the expression of proliferating cell nuclear antigen(PCNA)and cyclin D,thereby increasing the ratio of S phase cells in response to ZEAinduced apoptosis via phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)signaling pathway.Isorhamnetin also recovered(P<0.05)ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins(follicle-stimulating hormone receptor[FSHR]and cytochrome P450 family 19subfamily a member 1[CYP19A1]).Collectively,these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage,which promotes proliferation,alleviates apoptosis,endoplasmic reticulum stress,oxidative stress,and steroidogenesis disorder.

异鼠李素通过下调Notch1通路抑制HepG2细胞的增殖并诱导其凋亡

目的探究异鼠李素(Isorhamnetin,ISO)是否通过下调Notch1的表达来抑制肝癌HepG2细胞增殖并促进其凋亡。方法使用细胞毒性实验噻唑蓝(MTT)法检测评估HepG2细胞存活率,并使用流式细胞仪分析细胞的凋亡水平。克隆形成实验反应不同异鼠李素浓度下HepG2细胞的增殖情况。采用Western blot测定P53、Bcl-2和Bax的表达情况。结果处理ISO后,HepG2细胞的存活能力表现出明显的剂量和时间依赖性抑制。在ISO处理24 h后,HepG2细胞系中诱导了凋亡。ISO处理以及敲除Notch1蛋白后,上调抑癌基因P53的表达,上调细胞凋亡相关蛋白Bax的表达以及下调Bcl-2的表达。进一步实验发现,Notch1 siRNA可以增强ISO的抗肿瘤特性。结论ISO通过下调Notch1通路抑制肝细胞癌(HCC)细胞的增殖并促进其凋亡。

异鼠李素介导miR-454-3p/PIK3R1轴对口腔鳞状细胞癌细胞生物学特性的影响

目的:探讨异鼠李素(ISO)对口腔鳞状细胞癌(OSCC)细胞生物学特性的影响,以及在该过程中对miR-454-3p/PIK3R1轴的调控机制。方法:MTT法检测ISO对TSCC1细胞的毒性,随后将TSCC1细胞分为OSCC组、ISO组、ISO+miR-NC组、ISO+miR-454-3p mimic组。MTT法检测各组细胞的存活率;克隆形成实验检测各组细胞增殖情况;流式细胞术检测各组细胞凋亡;Transwell实验检测各组细胞迁移和侵袭能力;RT-qPCR法检测各组细胞中miR-454-3p和PIK3R1的mRNA水平;Western blotting法检测各组细胞中PIK3R1蛋白的表达;双荧光素酶实验验证miR-454-3p与PIK3R1的靶向关系;建立裸鼠移植瘤模型,将小鼠分为模型组、ISO组、ISO组+agomir-NC组,ISO组+miR-454-3p agomir组,每组5只,干预15 d后检测各组小鼠的肿瘤质量和体积;RT-qPCR法检测肿瘤组织中miR-454-3p和PIK3R1的mRNA水平。结果:体外实验发现,与OSCC组比较,ISO组和ISO+miR-NC组细胞存活率、迁移和侵袭细胞数量、miR-454-3p表达水平均降低(P<0.05),细胞凋亡率、PIK3R1 mRNA和蛋白表达水平均升高(P<0.05);miR-454-3p mimic回补实验逆转了ISO对OSCC的抑癌作用及PIK3R1的表达水平(P<0.05),同时双荧光素酶报告基因实验验证了miR-454-3p与PIK3R1之间存在靶向关系;体内实验发现,与模型组比较,ISO组小鼠移植瘤的质量和体积均减小(P<0.05),miR-454-3p表达水平、PIK3R1 mRNA表达水平以及miR-454-3p agomir的回补实验结果均与体外实验保持一致。结论:ISO能够通过调节miR-454-3p/PIK3R1轴抑制OSCC细胞的增殖、迁移和侵袭,促进细胞凋亡。

异鼠李素通过抑制LncRNA-gm33782减轻AKI肾小管炎性细胞凋亡

目的探讨长链非编码RNA-gm33782(LncRNA-gm33782)在急性肾损伤(acute kidney injury,AKI)肾小管损伤中的功能以及异鼠李素改善肾小管炎性细胞凋亡的作用机制。方法将48只雄性C57BL/6J小鼠随机分为对照组、模型组、治疗组(AKI模型构建后灌胃给予30 mg/kg异鼠李素)、空载质粒电转染组、LncRNA-gm33782敲低组、LncRNA-gm33782过表达组、LncRNA-gm33782敲低组+模型组、LncRNA-gm33782过表达+治疗组8组,通过腹腔一次性注射20 mg/kg顺铂诱导小鼠AKI,原位电转染技术用于介导肾LncRNA-gm33782的敲低和过表达。采用Chirp实验捕获AKI肾LncRNA-gm33782结合蛋白进行质谱分析,挖掘LncRNA-gm33782直接作用靶蛋白;通过观察电转染敲低LncRNA-gm33782和异鼠李素(isorhamnetin,ISO)治疗干预后小鼠肾功能、病理结构改变、肾炎性因子(IL-1β、IL-6、TNF-α)表达,评价LncRNA-gm33782在AKI中的作用;NF-κB被作为介导炎症的关键信号通路,Bax、Bcl-2蛋白表达量以及流式凋亡检测结果被用于评价异鼠李素对AKI肾小管炎性细胞凋亡的治疗作用。结果AKI小鼠肾表现出严重的肾小管损伤以及巨噬细胞浸润和炎症,异鼠李素的治疗干预和LncRNA-gm33782电转染敲低均能够减轻AKI肾损伤。LncRNA-gm33782主要表达于AKI损伤的肾小管细胞,质谱检测发现补体因子-H(complement factor-H,CFH)与其有直接结合关系。体外过表达LncRNA-gm33782后,CFH表达量随即升高,而异鼠李素对AKI细胞炎性凋亡的治疗作用受到抑制。结论异鼠李素是通过抑制LncRNA-gm33782对CFH的调控作用减轻AKI肾小管细胞炎性凋亡。

异鼠李素对口腔鳞状细胞癌细胞增殖和糖酵解影响的研究

为探讨异鼠李素(isorhamnetin)对人口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)(简称口腔鳞癌)细胞增殖以及糖酵解的影响,给予不同剂量异鼠李素处理口腔鳞癌细胞系CAL27和SCC9,并采用细胞活力分析和软琼脂集落实验检测异鼠李素对口腔鳞癌细胞增殖的影响;借助糖酵解分析实验检测异鼠李素对口腔鳞癌细胞乳酸产生和葡萄糖消耗的影响;利用蛋白质印迹法明确异鼠李素对口腔鳞癌细胞糖酵解相关信号通路的调控;最后,通过异种移植瘤模型验证异鼠李素在体内对肿瘤的影响。体外实验结果显示,异鼠李素以剂量依赖的方式抑制CAL27和SCC9细胞的增殖与糖酵解,并且这种抑制作用与糖酵解关键限速酶己糖激酶2(hexokinaseⅡ,HK2)而不是磷酸果糖激酶(phosphofructokinase,PFK)和丙酮酸激酶M2(pyruvate kinase M2,PKM2)的抑制有关。体内结果表明,异鼠李素抑制了口腔鳞癌在体内的生长,并降低了肿瘤组织中Ki67和HK2的表达。总的来说,研究结果表明,异鼠李素可抑制口腔鳞癌的增殖与糖酵解,且这种抑制效应与HK2表达降低有关。

基于HPLC指纹图谱的沙棘叶黄酮类功效成分研究

目的:采用高效液相色谱法(HPLC)建立不同产地沙棘叶指纹图谱,对其中异鼠李素、槲皮素和山柰酚含量进行测定,并对不同产地的沙棘叶品质进行综合评价。方法:指纹图谱采用C_(18)色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.4%磷酸水溶液,梯度洗脱,流速为0.8 mL·min^(-1),柱温为30℃,进样量为10μL,检测波长为280 nm;采用“中药色谱指纹图谱相似度评价系统”(2012版)软件进行指纹图谱相似度评价。含量测定采用C_(18)色谱柱(250 mm×4.6 mm,5μm),以甲醇-0.1%甲酸水溶液(58∶42)为流动相,流速为1.0 mL·min^(-1),柱温为30℃,进样量为10μL,检测波长为370 nm。结果:建立了不同产地沙棘叶HPLC指纹图谱,相似度为0.647~0.910。通过和对照品比对,对26个共有峰进行峰指认,其中9个共有峰鉴定为英国栎鞣花酸、沙棘宁B、木麻黄鞣宁、表儿茶素、水仙苷、表没食子儿茶素、芦丁、异槲皮苷和山柰酚-3-O-芸香糖苷;对不同产地沙棘叶中异鼠李素、槲皮素、山柰酚进行HPLC定量分析,结果表明,槲皮素、山柰酚、异鼠李素的质量分数分别为0.12%~0.33%、0.07%~0.32%、0.10%~0.49%。山西忻州沙棘叶槲皮素质量分数最高,为0.33%;内蒙古鄂尔多斯沙棘叶山柰酚质量分数最高,为0.32%;新疆塔城沙棘叶异鼠李素质量分数最高,为0.49%。结论:该方法精密度、稳定性、重复性良好,可有效评价沙棘叶品质。不同产地沙棘叶黄酮类功效成分含量差异较大。

一测多评法同时测定沙芪浓缩丸中5种黄酮

目的 建立一测多评法同时测定沙芪浓缩丸中毛蕊异黄酮苷、水仙苷、银锻苷、异鼠李素、芒柄花素的含量。方法 该药物90%甲醇(含1%甲酸)提取液的分析采用Welch LP-C18色谱柱(250 mm×4.6 mm, 5μm);流动相乙腈-0.4%磷酸,梯度洗脱;体积流量1.0 mL/min;柱温30℃;检测波长260 nm。以毛蕊异黄酮苷为内标,计算其他4种黄酮的相对校正因子,计算其含量。结果 5种黄酮在各自范围内线性关系良好(r≥0.999 9),平均加样回收率91.90%~99.82%,RSD 0.26%~0.64%。一测多评法所得结果与外标法接近(RAD<2%)。结论 该方法准确可行,可用于沙芪浓缩丸的质量控制。

异鼠李素通过调控TLR4/NF-κB信号通路对高氧诱导大鼠急性肺损伤的保护作用

目的:探讨异鼠李素对高氧诱导大鼠急性肺损伤(ALI)的保护作用及其可能机制。方法:32只SD大鼠随机分为正常对照组、模型组、异鼠李素组、异鼠李素+瑞沙托维组[瑞沙托维(TAK-242)为TLR4特异性抑制剂],每组8只,除正常对照组正常饲养外,其余各组大鼠均置于自制高氧箱中3 d构建ALI模型,模型构建成功后,按照各组给药剂量进行腹腔注射。模型组及正常对照组腹腔注射等体积生理盐水。各组注射用药均为1次/d,连续7 d。采集大鼠腹主动脉血、肺泡灌洗液、肺组织,血气分析仪测定大鼠氧分压(PaO_(2))、二氧化碳分压(PaCO_(2)),烘箱法测定大鼠肺组织湿重/干重(W/D),HE染色观察大鼠肺组织病理学变化,并对其进行肺组织损伤评分,免疫荧光染色法观察各组大鼠肺组织中TLR4表达水平,ELISA检测大鼠肺泡灌洗液中炎症因子水平,Western blot测定大鼠肺组织Toll样受体4/核因子κB(TLR4/NF-κB)通路相关蛋白表达情况。结果:与正常对照组相比,模型组大鼠PaO_(2)值显著降低,PaCO_(2)、W/D值显著升高,肺组织损伤严重,肺损伤评分、肺泡灌洗液中炎症因子水平、肺组织中TLR4/NF-κB通路相关蛋白表达显著升高;与模型组相比,异鼠李素组及异鼠李素+瑞沙托维组大鼠肺组织损伤得到缓解,PaO_(2)值显著升高,PaCO_(2)、W/D值、肺损伤评分、肺泡灌洗液中炎症因子水平、肺组织中TLR4/NF-κB通路相关蛋白表达显著降低;与异鼠李素组相比,异鼠李素+瑞沙托维组大鼠肺组织损伤得到改善,PaO_(2)值显著升高,PaCO_(2)、W/D值、肺损伤评分、肺泡灌洗液中炎症因子水平、肺组织中TLR4/NF-κB通路相关蛋白表达显著降低。结论:异鼠李素能缓解高氧诱导的大鼠ALI,其机制可能与抑制TLR4/NF-κB通路有关。